One of the earliest responses to 12-0-tetradecanoylphorbol-13-acetate (TPA) may be the generation of reactive oxygen species. Therefore, antioxidants with their capacity to terminate free radical chain reactions would be expected to modify the tumor promotion process. Recent studies from our laboratory indicate that TPA decreases rapidly the intracellular ratio of reduced glutathione (GSH)/oxidized glutathione (GSSG) in epidermal cell suspensions. Moreover, treatments with cysteine (Cys), GSH and Alpha-tocopherol inhibit dramatically the induction of epidermal ornithine decarboxylase (ODC) activity by TPA, one of the essential components of mouse skin tumor promotion, as well as the incidence of skin tumors in the initiation-promotion protocol. Our findings suggest that Cys and GSH might inhibit TPA-induced ODC activity and the promotion of skin papillomas through an increase in the intracellular ratio of GSH/GSSG. Since the inhibitory effects of Cys on both the decrease in the ratio of GSH/GSSG and the induction of ODC activity by TPA are greatly reduced by the inhibitors of Gamma-glutamyl transpeptidase (Gamma-GT) and Gamma-glutamylcysteine synthetase, we believe it is important to rigorously study the metabolism of GSH, a natural antioxidant which inhibits chemical carcinogenesis, during skin tumor promotion. First, we will study the effects of various tumor promoters on GSH level, Gamma-GT and GSH peroxidase activities, and O2 production in isolated epidermal cells. Second, because functional interrelationships between sulfur-containing amino acids, selenium, vitamin E, and GSH peroxidase have been postulated, we will determine if treatments including GSH level-raising agents, selenoamino acids and Alpha-tocopherol might increase the antioxidant GSH peroxidase protective system of the cells and inhibit the oxidative challenge linked to skin tumor promotion. Finally, we will determine the effectiveness of combined treatments with GSH level-raising agents, selenium-containing compounds and Alpha-tocopherol as modifiers of the effects of TPA on GSH metabolism, stimulators of the GSH-dependent antioxidant protective system and inhibitors of multistage skin tumor promotion. These studies should help us to identify new inhibitors of skin carcinogenesis and in general should provide a better understanding of the biochemistry of tumor promotion and its regulation.